Japanese Encephalitis Virus in Meningitis Patients, Japan

Cerebrospinal fluid specimens from 57 patients diagnosed with meningitis were tested for Japanese encephalitis virus. Total RNA was extracted from the specimens and amplified. Two products had highest homology with Nakayama strain and 2 with Ishikawa strain. Results suggest that Japanese encephalitis virus causes some aseptic meningitis in Japan.


The Study
CSF specimens were obtained for diagnostic purposes from 170 patients with a clinical diagnosis of aseptic meningitis from August to October in each year from 1999 to 2002, in Hiroshima prefecture. These CSF specimens were sent to the Division of Microbiology II, Hiroshima Prefectural Institute of Health and Environment, for viro-logic examination. Viruses were isolated from 112 of 170 CSF specimens in cell cultures by using Vero cells, BGM cells, FL cells, Hep2 cells, and RD18S cells. Enteroviruses and mumps viruses were isolated from 96 and 16 CSF specimens, respectively (see online Appendix Table, available from http://www.cdc.gov/ncidod/eid/04-0285_app .htm). Thus, etiologic agents were not determined for 58 meningitis cases. CSF specimens from 57 of these 58 cases were used to detect Japanese encephalitis virus genome. Whether the 57 patients had been vaccinated for Japanese encephalitis virus was not known.
Total RNA was extracted from CSF by using ISOGEN-LS (Nippon Gene, Tokyo, Japan). The RNA pellet was resuspended in DEPC Treated Water (Invitrogen Corp., Carlsbad, CA, USA). The RNA was reverse transcribed and amplified by using polymerase chain reaction (PCR) with AMV Reverse Transcriptase XL (Life Sciences Inc., St. Petersburg, Florida, USA.) and Tth DNA Polymerase (Toyobo Co., Ltd., Osaka, Japan) with the primer pair reported by Morita et al. (5). The PCR product was nested PCR-amplified by TaKaRa EX Taq (Takara Bio Inc., Otsu, Japan), with the primer pairs for E gene region reported by Kimura  Electrophoresis demonstrated the band with expected size of 326 bp in 4 of 57 PCR products ( Figure 1). Two (numbers 1 and 2) of these products were sequenced on 326 bp and 247 bp, respectively, and the highest homology was with Japanese encephalitis virus, Nakayama strain (genotype III). PCR products 3 and 4 were sequenced on 121 bp and 187 bp by using other primers, and the highest homology was with Japanese encephalitis virus, Ishikawa strain (genotype I) ( Table 1).
Patients 1-4 lived in Hiroshima prefecture ( Figure 2). Patient 1 was a 3-year-old boy and patient 4 was a 6-yearold girl ( Table 2). They became sick in early August 2000. Patient 2 was a 2-year-old girl who became sick in late August 2000. Patient 3 was a 4-year-old boy who got sick in mid-September 2000. CSF samples were obtained 2-3 days after the onset of illness. All 4 patients had symptoms characteristic of meningitis and were clinically diagnosed with meningitis.

Conclusions
Enteroviruses are the most frequent cause of aseptic meningitis in summer and autumn in Japan (online Appendix Table). In the CSF samples from these 4 patients, however, enteroviruses were not found. Watt et al. reported that in Thailand, 14% of adult patients with acute, undifferentiated fever had a diagnosis of Japanese encephalitis virus infection because anti-Japanese encephalitis virus immunoglobulin (Ig) M antibodies were found (10). Many physicians in Japan consider Japanese encephalitis virus to be mainly associated with encephalitis, and examination for the virus is not usually conducted for patients with meningitis or undifferentiated fevers.
Three Japanese encephalitis cases occurred in 2002 for the first time in 12 years, but anti-Japanese encephalitis virus IgM antibodies were detected in porcine serum samples every year in Hiroshima prefecture (data not shown). So we expected that Japanese encephalitis virus might be associated with aseptic meningitis, and CSF specimens from 57 cases during the past 5 years (1998-2002) had been stored at -30°C in Hiroshima Prefectural Institute of Health and Environment. Those specimens were used to detect Japanese encephalitis virus genome retrospectively. Four of 57 CSF samples were Japanese encephalitis virus genome-positive by nested PCR. We may find more